title: Simultaneous isolation of CD8(+) and CD4(+) T cells specific for multiple viruses for broad antiviral immune reconstitution after allogeneic stem cell transplantation.
abstract: Opportunistic viral infections can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation. Clinical studies have shown that adoptive transfer of donor-derived T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV), or human adenovirus (HAdV) can be a safe and effective treatment of infections with these major viral pathogens. The aim of this study was to develop a method for the simultaneous isolation of coordinated CD8(+) and CD4(+) memory T-cell responses against a broad repertoire of viral epitopes. To ensure that the method was applicable to a wide variety of virus-specific T cells that may differ in phenotypic and functional properties, we focused on T cells specific for the persistent viruses, CMV and EBV, and T cells specific for HAdV and influenza (FLU), which are not repetitively activated in vivo after initial viral clearance. Following in vitro activation, nearly all T cells specific for these viruses produced interferon γ (IFN-γ) and tumor necrosis factor α, and expressed CD137, whereas the populations varied in the production of interleukin-2, degranulation, and expression of phenotypic markers. Different kinetics of IFN-γ production were observed in CMV/EBV-specific T cells and HAdV/FLU-specific T cells. However, after the stimulation of peripheral blood from seropositive donors with viral protein-spanning peptide pools, the activated virus-specific CD8(+) and CD4(+) T cells could be simultaneously isolated by either IFN-γ-based or CD137-based enrichment. This study provides an efficient and widely applicable strategy for the isolation of virus-specific T cells, which may be used for the reconstitution of virus-specific immunity in allogeneic stem cell transplantation recipients.
authors.affiliation: Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands. m.l.zandvliet@lumc.nl
authors.forename: Maarten L
authors.initials: ML
authors.lastname: Zandvliet
authors.forename: Ellis
authors.initials: E
authors.lastname: van Liempt
authors.forename: Inge
authors.initials: I
authors.lastname: Jedema
authors.forename: Simone
authors.initials: S
authors.lastname: Kruithof
authors.forename: Michel G D
authors.initials: MG
authors.lastname: Kester
authors.forename: Henk-Jan
authors.initials: HJ
authors.lastname: Guchelaar
authors.forename: J H Frederik
authors.initials: JH
authors.lastname: Falkenburg
authors.forename: Pauline
authors.initials: P
authors.lastname: Meij
mesh_terms: D056704:Adaptive Immunity; D000260:Adenoviruses, Human / Q000276:immunology; D019264:Adoptive Transfer; D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000637:transplantation* / Q000821:virology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000637:transplantation* / Q000821:virology; D003587:Cytomegalovirus / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000276:immunology; D006801:Humans; D016867:Immunocompromised Host; D007156:Immunologic Memory; D018189:Immunomagnetic Separation / Q000379:methods*; D007371:Interferon-gamma / Q000276:immunology; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009894:Opportunistic Infections / Q000276:immunology* / Q000821:virology; D009975:Orthomyxoviridae / Q000276:immunology; D033581:Stem Cell Transplantation; D014184:Transplantation, Homologous; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma